Process for the physical depolymerization of glycosaminogylcanes and products obtained thereform

ABSTRACT

The invention relates to a process for the depolymerization of glycosaminoglycanes characterized by the use of electron beam radiation, optionally in the presence of an organic compound selected from the group consisting of ethers, alcohols, aldehydes, amides and formic acid. The invention also relates to the intermediate depolymerized heparin obtained by the process. The intermediate depolymerized heparin can be dissolved in a buffer solution and fractionated by gel permeation for obtaining the desired molecular weight.

This application is a United States national filing under 35 U.S.C. §371of international (PCT) application No. PCT/EP03/006446, filed Jun. 18,2003, designating the US, and claiming priority to Italian ApplicationNo. MI2002A001372, filed Jun. 21, 2002.

STATE OF THE ART

Glycosaminoglycanes are natural products of large pharmaceuticalinterest. Among the most widely used we can mention heparin, dermatan,heparansulphate and chondroitines.

The molecular weight of the natural products varies considerably andnormally ranges from 5 to 40 kDa. It is however known that for certainapplications lower molecular weights lead to higher pharmacologicalactivity. The high molecular weight of these compounds often rendersimpossible their oral administration. Furthermore, it is known thatspecific activities of glycosaminoglycanes are related to relativelyshort saccharide sequences. Thus, it would be very advantageous todepolymerize glycosaminoglycanes reducing the molecular weight withoutloosing the active sites present in the molecule.

The chemical depolymerization of glycosaminoglycanes is well known inthe art and leads to glycosaminoglycanes of lower MW but also with alower S content.

EP 394 971 discloses an enzymatic or chemical depolymerization process.The obtained heparin oligomers have a sulphur content lower than 9%.

WO 90/04607 discloses a depolymerization of heparin and dermatansulfateby the use of H₂O₂ and Cu²⁺. The ratio SO₃ ⁻/COO⁻ is slightly lower thanin the starting heparin.

U.S. Pat. No. 4,987,222 discloses a method for the depolymerization ofheparin by the use of gamma rays. The examples disclose the preparationof heparin of Mw around 5,000 Da and with a high S content.

SUMMARY OF THE INVENTION

The present invention relates to a physical process for thedepolymerization of glycosaminoglycanes by the use of electron-beamradiation (EB). It also relates to the glycosaminoglycanes obtained bythis process.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a physical depolymerization process whichreduces the molecular weight of glycosaminoglycanes withoutsubstantially modifying the chemical structure of the same.

The objective is achieved through use of electron-beam radiation. Whenusing heparin as a starting material, this process results in a low toultra-low molecular weight heparin characterized by high S content.

The starting materials to be used in the process according to thepresent invention are natural glycosaminoglycanes such as heparin,heparansulphate, dermatane and chondroitine. Other suitable startingmaterials are derivatives of glycosaminoglycanes obtained by knownmethods. Thus, for example, the N-acetyl or N-sulphate groups of theresidues of hexosamine can be transformed into amino groups throughN-desulphation or N-deacetylation reactions and the sulphate groups ofthe uronic acids can give rise to epoxy groups through desulphationreactions.

In another embodiment, it is possible to use as a starting material forthe process of the present invention a glycosaminoglycane which hasalready undergone a depolymerization process either chemical orenzymatic. The use of partly depolymerized glycosaminoglycanes is forexample relevant in case of heparin which has undergone an acidpretreatment that has as a side effect partial depolymerization, or whendepolymerizing functionalized glycosaminoglycanes. The conditions usedfor the introduction of functional groups are sometimes also causingreduction of the molecular weight of the polysaccharide.

Thus, not only it is possible to perform both steps by usingelectron-beam radiation, but it is possible to perform a firstdepolymerization step by using electron-beam radiation followed by asecond step using chemical-enzymatic depolymerization, or to perform afirst step of chemical-enzymatic depolymerization followed byelectron-beam radiation depolymerization.

The process of the present invention allows reduction of the molecularweight of the glycosaminoglycane without sensible modification of thechemical structure of the polysaccharide.

The dose of radiation used in the depolymerization process depends onseveral factors, e.g. the type of glycosaminoglycanes, the desired finalMw, the energy of the radiation. In general, the dose of radiation willvary in the range 400-8,000 kGy, preferably 800-6,000 kGy, morepreferably 1,200-5,000 kGy.

Preferably, the electron-beam radiation has an energy comprised between100 and 1000 keV, most preferably between 100 and 500 keV.

The depolymerization process can be performed in a broad range oftemperature, it is however preferred to maintain the temperature between0 and 50° C., most preferably between 20 and 40° C.

The depolymerization process according to the invention is preferablyperformed in aqueous solution, optionally in the presence of an organiccompound selected from the group consisting of alcohols, ethers,aldehydes, amides and formic acid. Preferably, the organic compound isselected from compounds of formula I, II and III.

wherein each R is independently selected from the group consisting of H,OH, CHO, C₁-C₆ alkyl and acyl, optionally substituted by oxygen atoms;two R groups optionally join together to form a ring.

Preferred examples of alcohols are: methanol, ethanol, n-propanol,isopropanol, n-butanol, isobutanol, glycerol.

Preferred examples of ethers are: tetrahydrofurane, dioxane,diethylether, tertbutylmethylether, dioxolane.

Examples of aldehydes are formaldehyde, glyoxal, acetaldehyde orstabilized forms thereof (trioxane, glyoxal trimeric dihydrate).

Preferred examples of amides are: N,N-dimethylformamide,N,N-dimethylacetamide, N,N-diethylformamide, N-methylpyrrolidone.

The concentration of glycosaminoglycane in the solution to be submittedto radiation can vary in a broad range. Preferably it is comprisedbetween 2 and 25% w/v, more preferably between 5 and 15%.

After irradiation, the solutions are optionally discolored either byusing an oxidizing treatment or by passing them on proper resins. Thesolution is then generally purified by precipitation in hydrophilicsolvent. The obtained paste can be redissolved in water and lyophilizedby vacuum distillation.

It is also possible to fractionate the intermediate depolymerizedglycosaminoglycane by Gel Permeation Chromatography. The fractionscontaining the desired molecular weights are collected, concentrated byultra filtration and lyophilized.

The process of the present invention is preferably performed by using adynamic irradiation process.

With the term “dynamic irradiation process” it is meant a processwherein the irradiation is performed on a thin layer of liquid which isfluxing in front of the electron-beam window. In this way, theefficiency of the irradiation process is increased.

The process can be performed either in batch or in continuous mode. Theapparatus is preferably formed of a reservoir from which the liquidmoves to the irradiation area. The liquid is then returned to thereservoir.

The exposure of the solution to the electron stream can take place indifferent ways:

-   -   in front of the window an inclined plane is placed, on which a        thin layer of solution flows,    -   in front of the window can be placed a system of thin pipes        which allow the exposition of the solution to the electrons,    -   the solution can flow directly on the window.

The optimal conditions of irradiation are determined through preliminarydosimetry.

The dosimetry has been performed considering the typical conditions ofirradiation of the solution in terms of:

-   -   a) properties relating to the beam of electrons, i.e.        -   beam energy (measured in keV)        -   beam current (measured in mA);    -   b) properties relating to the geometry of the irradiation, i.e.        -   distance beam source-solution to process,        -   presence of possible shields or other nearby material that            can be source of secondary radiation.

The dosimetry is in any case performed for a limited period of time,since the dose administered to the material is directly proportional tothe time of the exposition and is determined in static conditions, whilein reality the process is dynamic.

Experimental Section Characterization of the Products

Molecular weight (Mw) was determined by size exclusion chromatography(European Pharmacopoeia 4^(th) ed.: 2.2.30 e 2.2.46 for chromatographictechniques and 01/2002:0828 p. 1297 for method).

β-rays Irradiation

The solution irradiation process takes place inside an electron-beamapparatus.

The beam is generated by a hot cathode, constituted of a tungstenfilament to whom a high voltage is applied.

The beam generation area is posed under vacuum. Such a vacuum isobtained by the combined action of two pumps, a mechanical one and aturbomolecular one.

The aspiration generated by these two pumps allows the achievement ofideal conditions for the free circulation of electrons which otherwisewould be slowed down by the air present around the cathode.

The beam reaches the region outside the chamber where it is generatedpassing through a very thin titanium film (thickness 10 μm). By theirpassage X rays are also generated. The solution to be irradiated isplaced immediately outside this titanium film, at a distanceconveniently as small as possible so that the beam exiting the film isnot significantly attenuated and thus the use optimised without uselesswastes.

The solution to be irradiated is circulated in proximity of the windowsfrom where the beam exits and it is directly exposed to it. Thecirculation circuit is provided with an external pumping system. Thesolution is continuously circulated inside and outside the shielded areaand therefore can be regularly sampled and fresh solution for processingcan be added.

EXAMPLE 1

One liter of 10% sodium heparin solution, free of heavy metal wasprepared. The solution was transferred to an electron beam apparatus andthe circulation was started in mobile descending phase, over porousglass wool tissue of 1 mm thickness, with a flow rate of 101/h by usinga peristaltic pump.

Starting the EB irradiation at 5 mA and 300 keV, the cooling system wasactivated in order to maintain the temperature between 25 and 35° C. Thedepolymerization was monitored by collecting samples, at fixedintervals, and the molecular weight of the composition was determined.The variation in time is shown in Table 1.

The electron beam was stopped and the collected solution was thenspray-dried to obtain the intermediate product which was fractionated bygel permeation.

TABLE 1 Minutes >10.000 Da kGy Mw 0 30% — 8.364 5 17% 134 5.941 10 12%268 5.050 15 9% 402 4.523 30 4% 804 3.682 45 2% 1.206 3.240 60 1% 1.6083.014

EXAMPLE 2

The example was conducted under the identical conditions of example 1,but with an intensity of current of 10 mA.

At the end, the electron beam is stopped and the collected solutionundergoes spray-drying to obtain the intermediate product which isfractionated by Gel Permeation

TABLE 2 Minutes >10.000 kGy Mw 0 30% — 8.364 5 12% 268 4.888 10 7% 5364.053 15 4% 804 3.526 30 2% 1.608 3.040 45 1% 2.412 2.852 60 — 3.2162.716

EXAMPLE 3

The example was conducted under the identical conditions of example 1,but with a beam energy of 150 keV and a current of 5 mA. The results arereported in Table 3.

TABLE 3 Minutes >10.000 kGy Mw 0 30% — 8.364 5 24% 161 7163 10 21% 3226542 15 20% 483 6337 30 17% 966 5968 45 16% 1449 6333 60 13% 1932 568175 10% 2415 5235 90 8% 2898 4806

EXAMPLE 4

The example was conducted under the identical condition of example 1,but with the addition of 0.4% v/v of isopropanol. Table 4 reports theobtained results.

TABLE 4 Minutes >10.000 Da kGy Mw 0 30% — 8.364 5 20% 134 6265 10 16%268 5653 15 12% 402 4851 30 5% 804 3760 45 3% 1.206 3298 60 1% 1.6083018 75 1% 2010 2855 80 — 2144 2780

EXAMPLE 5

The example was conducted under the identical condition of example 2,but with the addition of 0.4% v/v of isopropanol. Table 5 reports theobtained results.

TABLE 5 Minutes >10.000 Da kGy Mw 0 30% — 8.364 5 16% 268 5625 10 10%536 4626 15 7% 804 4043 20 4% 1072 3559 25 3% 1.340 3289 30 3% 1.6083261 45 1% 2412 2913 55 1% 2948 2921

1. A process for the depolymerization of glycosaminoglycanes consistingessentially of: exposing an aqueous solution to electron beam radiation,said solution consisting essentially of: i) water; ii) aglycosaminoglycane; and iii) optionally, an organic compound selectedfrom the group consisting of Formula I, Formula II, and Formula III:

wherein each R is independently selected from the group consisting of H,OH, CHO, C₁-C₆ alkyl and acyl, optionally substituted by oxygen atoms;two R groups optionally join together to form a ring, and wherein theconcentration of the organic compound is from 0.1% to 5%.
 2. The processaccording to claim 1, wherein said exposing step is performed using adynamic irradiation process.
 3. The process according to claim 1,wherein the glycosaminoglycane is heparin.
 4. The process according toclaim 1, wherein the electron-beam radiation has an energy of from 100keV to 1000 keV.
 5. The process according to claim 1, wherein theorganic compound is selected from the group consisting of methanol,ethanol, n-propanol, isopropanol, n-butanol, isobutanol, glycerol,tetrahydrofurane, dioxane, diethylether, tertbutylmethylether,dioxolane, formaldehyde, glyoxal, acetaldehyde, N,N-dimethyl formamide,N,N-dimethylacetamide, N,N-diethylformamide, and N-methylpyrrolidone. 6.The process according to claim 1, wherein the amount of radiation usedis from 400 kGy to 8,000 kGy.